The group-specific component (Gc) protein of human serum was first noted because of its electrophoretic polymorphisms between individuals. It is a 58,000 dalton glycoprotein produced by the liver. The worldwide distribution of polymorphisms and variants of this protein has been well studied. Two biological activities of this protein have been recently defined: it serves as a serum transport protein for vitamin D and its metabolites (and is therefore known as vitamin D binding protein or DBP), it binds to actin monomers preventing their polymerization. This project involves the study of the gene(s) encoding this polymorphic and bifunctional protein. The project's specific aims are to isolate and sequence a cDNA clone complementary to DBP mRNA, isolate and sequence the gene or genes encoding DBP, confirm their chromosomal location, and transfer the isolated gene to cells in tissue culture in order to study its effect on the cytoskeleton and vitamin D metabolism. The cDNA will be isolated from a library of human liver cDNA clones by three complementary techniques: a positive translational assay, in situ hybridization with cDNA probe to immunoabsorbed DBP mRNA, and positive/negative hybridization with a cDNA probe to total mRNA from Hep 3B or Hep G2 liver cells in culture, respectively. The gene will be isolated from a chromosomal DNA library by in situ hybridization with the isolated DBP cDNA. DNA sequencing will be done utilizing both the Maxam-Gilbert methodology and a non-random dideoxy chain termination technique. The binding regions for 25(OH)D and actin within the DBP molecule will be delineated by generating exon deletion and oligonucleotide-directed mutations in the gene, expressing these mutant genes inserted in bovine papilloma virus in its host C127 cells, and assaying the culture media for 3H 25(OH)D and 125I-actin binding. The effect of DBP on the actin cytoskeleton will be studied by introduction into cells of the DBP gene to which an inducible promoter has been ligated. These studies are designed to establish a firm biochemical foundation for the study of DBP, delineate its biological roles, and define pathologic states related to its absence or genetic variation.